Methods

ABSTRACT

A method for promoting entry of an agent (introduced agent) into a cell, the method comprising the step of complexing the introduced agent in the presence of an entry-promoting agent and then exposing to cells, wherein the entry-promoting agent comprises a linear and/or branched or cyclic polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof according to the following Formula 1a &amp; b. The method also provides a means for formation of nanoparticles formed between the entry promoting agent and the introduced agent. wherein: “n”, refers to number of repeating units in the polymer, and n can vary from 2 to 1000, for example from 2 or 5 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 or 900; G 1  and G 2  independently represent a cationic group comprising biguanide or guanidine, wherein L 1 , and L 2  are directly joined to a Nitrogen atom of the guanide; L 1  and L 2  are linking groups between the G 1  and G 2  cationic groups in the polymer and independently represent an aliphatic group containing C 1 -C 40  carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C 4 , C 5 , C 6 , C 7 , C 8 , C 9  or C 10 ; C 1 -C 10 , -C 20 , -C 30 , -C 40 , -C 50  -C 60  -C 70 , -C 80 , -C 9 0, -C- 100 , -C 110 , -C 120 , -C 130  or -C 140 , alkyl; or a C 1 -C 140  (for example C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9  or C 10 ; C 1 -C 10 , -C 20 , -C 30 , -C 40 , -C 50  -C 60 , -C 70 , -C 80 , -C 90 , -C 100 , -C 11 0, -C 120 , -C 130 o or -C 140 ), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, or oxyalkylene radical; or a polyalkylene radical optionally interrupted by one or more, preferably one, oxygen, nitrogen or sulphur atoms, functional groups or saturated or unsaturated cyclic moiety; N and G 3  are optional end groups; X can be either present or absent; L 3 , L 4  and X are linking groups between the G 4  and G 5  cationic groups in the polymer and independently represent an aliphatic group containing C 1 -C 140  carbon atoms, for example an alkyl group such as methylene, ethylene, propylene, C 4 , C 5 , C 6 , C 7 , C 8 , C 9  or C 10 ; C 1 -C 10 , -C 20 , -C 30 , -C 40 , -C 50  -C 60 , -C 70 , -C 80 , -C 90 , -C 100 , -C 110 , -C 120 , -C 130  or -C 140 , alkyl; or L 3  and L 4  and X can independently be C 1 -C 140  (for example C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9  or C 10 ; C 1 -C 10 , -C 20 , -C 30 , -C 40 , -C 50  -C 60 , -C 70 , -C 80 , -C 90 , -C 100 , -C 110 , -C 120 , -C 130  or -C 140 ), cycloaliphatic, heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, oxyalkylene radicals; or a polyalkylene radical optionally interrupted by one or more, preferably one, oxygen, nitrogen or sulphur atoms, functional groups as well as saturated or unsaturated cyclic moiety; “G 4 ” and “G 5 ” are cationic moieties and can be same or different, and at least one of them is a biguanidine moiety or carbamoylguanidine, and the other moiety may be biguanidine or carbamoylguanidine or amine; and cationic moieties G 4  and G 5  do not contain single guanidine groups. The entry-promoting agent may comprise homogeneous or heterogeneous mixture of one or more of agents arising from formulae 1 a and b, for example polyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide (PTMB), polyethylene hexamethylene biguanide (PEHMB), polymethylene biguanides (PMB), poly(allylbiguanidnio-co-allylamine), poly(N-vinylbiguanide), polyallybiguanide.

METHODS

The present invention relates to the field of methods and reagents forpromoting entry of an agent, for example a nucleic acid, into a cell.

Nucleic acids and analogues thereof have many applications in basicresearch and both prophylactic and therapeutic interventions. However,prokaryotic and eukaryotic cells are in general impermeable to nucleicacids/analogues. Typically nucleic acid/analogue uptake has requireddelivery vehicles, either viral or non-viral, in order to reach theirpotential. To be effective, the delivery vehicle/nucleic acid oranalogue combination or complex must overcome many challenges, forexample extracellular degradation, cell membrane barrier penetration andintracellular release, whilst minimising cytotoxicity and antigenicity.Many polypeptides (for example proteins, peptides) and other bioactivemolecules also are cell impermeable, but otherwise useful. For examplethe activity of many compounds in biochemical assays is much higher thanin cellular assays or in vivo. The drop off in cellular activity isbelieved to be largely due to poor delivery into cells.

Lipofectamine is a widely used example of an agent used in promotingentry of agents, for example nucleic acids, into cells. However,lipofectamine is considered to have toxic effects on mammalian cells inparticular, which can, for example, make in vitro experiments difficultto assess and in vivo experiments difficult to perform.

Other proposed entry-promoting agents are described in, for example, WO2009/015143 (Biodegradable cationic polymer gene transfer compositionsand methods of use); WO 02/22174 (cationic lipopolymer as biocompatiblegene delivery agent); WO2010/086406 (non-viral transfection agent); U.S.Pat. No. 5,958,894 (amphiphilic biguanide derivatives).

There is a need for alternative entry-promoting agents, for example fornucleic acids and other substances where activities are limited by poorcell entry. The present invention identifies entry-promoting agents andmethods. Such entry-promoting agents and methods are considered to bebeneficial, for example in providing good entry promotion, poorantigenicity, low cost and/or low toxicity, for example particularly foreukaryotic cells. Such entry-promoting agents and methods may, forexample, be useful in areas such as nucleic acid/analogue transfection,for example functional studies; generation of stable cell lines; genesilencing; and DNA vaccination. For example, such entry-promoting agentsand methods may, for example, be useful in relation to RNA interference(RNAi) or other antisense technologies, as will readily be appreciatedby those skilled in the art. The challenge of delivery of reagents ordrugs into cells is not limited to only nucleic acids. Proteins andpeptides also enter cells very poorly in general and entry-promotingtechnology would be useful. Also, many molecules that are described as“small molecules” being less than 1000 grams/mol enter cells poorly andtheir usefulness as reagents or drugs would be enhance by improved celldelivery technology.

PHMB (polyhexamethylene biguanide) is known as a safe and effectivebiocidal agent and is used as a sanitizer and preservative: U.S. Pat.No. 7,897,553, U.S. Pat. No. 4,758,595, US2008261841; US 20040009144.The present inventors have surprisingly found that PHMB and relatedmolecules are useful entry-promoting agents. It was surprisinglyobserved that PHMB (for example) itself enters a wide range of cells,including bacteria, fungi and mammalian cells. More surprisingly, PHMB(for example) is able to form nanoparticles with a wide range ofmolecules and deliver these molecules into such cells. Finally thedelivered molecules ranging from nucleic acids to small molecules werefound to be functional inside cells.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

A first aspect of the invention provides a method for promoting entry ofan agent into a cell, the method comprising the step of exposing thecell to the introduced agent in the presence of a polymer, wherein thepolymer comprises a linear and/or branchedpolymonoguanide/polyguanidine, polybiguanide, analogue or derivativethereof, for example according to the following formula 1a or formula1b, with examples given in tables 1 and 2, below:

wherein:

“n”, refers to number of repeating units in the polymer, and n can varyfrom 2 to 1000, for example from 2 or 5 to 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 or900;

G₁ and G₂ independently represent a cationic group comprising biguanideor guanidine, wherein L₁ and L₂ are directly joined to a Nitrogen atomof the guanide. Thus, the biguanide or guanidine groups are integral tothe polymer backbone. The biguanide or guanidine groups are not sidechain moieties in formula 1a.

Example of Cationic Groups:

In the present invention, L₁ and L₂ are the linking groups between theG₁ and G₂ cationic groups in the polymer. L₁ and L₂ can independentlyrepresent an aliphatic group containing C₁-C₁₄₀ carbon atoms, forexample an alkyl group such as methylene, ethylene, propylene, C₄, C₅,C₆, C₇, C₈, C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀, -C₇₀, -C₈₀,-C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀, alkyl; or L₁ and L₂ can(independently) be C₁-C₁₄₀ (for example C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈,C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀, -C₇₀, -C₈₀, -C₉₀, -C₁₀₀,-C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀) cycloaliphatic, heterocyclic, aromatic,aryl, alkylaryl, arylalkyl, oxyalkylene radicals, or L₁ and L₂ can(independently) be a polyalkylene radical optionally interrupted by oneor more, preferably one, oxygen, nitrogen or sulphur atoms, functionalgroups as well as saturated or unsaturated cyclic moiety. Examples ofsuitable L₁ and L₂ are groups are listed in table 1.

L₁, L₂, G₁ and G₂ may have been modified using aliphatic,cycloaliphatic, heterocyclic, aryl, alkaryl, and oxyalkylene radicals.

N and G₃ are preferably end groups. Typically the polymers of use in theinvention have terminal amino (N) and cyanoguanidine (G₃) or guanidine(G₃) end groups. Such end groups may be modified (for example with1,6-diaminohexane, 1,6 di(cyanoguanidino)hexane, 1,6-diguanidinohexane,4-guanidinobutyric acid) by linkage to aliphatic, cycloaliphaticheterocyclic, heterocyclic, aryl, alkylaryl, arylalkyl, oxyalkyleneradicals. In addition, end groups may be modified by linkage to receptorligands, dextrans, cyclodextrins, fatty acids or fatty acid derivatives,cholesterol or cholesterol derivatives or polyethylene glycol (PEG).Optionally, the polymer can end with guanidine or biguanide orcyanoamine or amine or cyanoguanidine at N and G₃ positions orcyanoamine at N and cyanoguanidine at G₃ position or guanidine at N andCyanoguanidne at G₃ positions or L1 amine at G3 and cyanoguanidine at N.G3 can be L₁-amine, L₂-cyanoguanidine or L₂-guanidine. Depending on thenumber of polymerization (n) or polymer chain breakage and sidereactions during synthesis, heterogeneous mixture of end groups canarise as described above as an example. Thus, the N and G3 groups can beinterchanged/present as a heterogeneous mixture, as noted above.Alternatively N and G₃ may be absent and the polymer may be cyclic, inwhich case the respective terminal L₁ and G₂ groups are linked directlyto one another.

In formula 1b, X can be either present or absent. L₃, L₄ and X are asnoted above for “L₁ or L₂”. Ir

Thus, L₃ and L₄ and X are the linking groups between the G₄ and G₅cationic groups in the polymer. L₃ and L₄ and X can independentlyrepresent an aliphatic group containing C₁-C₁₄₀ carbon atoms, forexample an alkyl group such as methylene, ethylene, propylene, C₄, C₅,C₆, C₇, C₈, C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀, -C₇₀, -C₈₀,-C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀, alkyl; or L₃ and L₄ and X canindependently be C₁-C₁₄₀ (for example C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉or C₁₀, C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀, -C₇₀, -C₈₀, -C₉₀, -C₁₀₀,-C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀), cycloaliphatic, heterocyclic, aromatic,aryl, alkylaryl, arylalkyl, oxyalkylene radicals, or L₃ and L₄ and X canindependently be a polyalkylene radical optionally interrupted by one ormore preferably one, oxygen, nitrogen or sulphur atoms, functionalgroups as well as saturated or unsaturated cyclic moiety. Examples ofsuitable L₃ and L₄ and X are groups are listed in table 2.

“G₄” and “G₅” are cationic moieties and can be same or different. Atleast one of them is a biguanidine moiety or carbamoylguanidine, and theother moiety may be as above (biguanidine or carbamoylguanidine) oramine. For the avoidance of doubt, in formula 1b, cationic moiety G₄ andG₅ do not contain only single guanidine groups. For example, G₄ and G₅typically do not contain single guanidine groups. Examples of suchcompounds are polyallylbiguanide poly(allylbiguanidnio-co-allylamine),poly(allylcarbamoylguanidino-co-allylamine), polyvinylbiguanide, aslisted in table 2.

Example of polyallylbiguanide is as shown below

In case of polyallylbigunidine L₃ and L₄ are identical, G₄ and G5 aresimilar, thus polyallylbiguanide can be simplified as below.

Example of poly(allylcarbamoylguanidnio-co-allylamine) is as shown below

The polymers for use in the invention will generally have counter ionsassociated with them. Suitable counter ions include but are not limitedto the following: halide (for example chloride), phosphate, lactate,phosphonate, sulfonate, amino carboxylate, carboxylate, hydroxycarboxylate, organophosphate, organophosphonate, organosulformate andorganosuflate.

Polymers for use in the invention can be either heterogeneous mixturesof polymers of different “n” number or homogenous fractions comprisingspecified “n” numbers purified by standard purification methods. Asindicated above the polymers may also be cyclic and in addition may bebranched.

Preferred numbers for “n” include 2-250, 2-100, 2-80 and 2-50.

TABLE 1 Examples of polymer analogues arising from formula 1a. Name L₁G₁ L₂ G₂ Polyhexamethylene biguanide (CH₂)₆ Biguanide (CH₂)₆ Biguanide(PHMB) Polyethylene biguanide (PEB) (CH₂)₂ Biguanide (CH₂)₂ BiguanidePolyethylenetetramethylene (CH₂)₂ Biguanide (CH₂)₄ Biguanide biguanidePolyethylene hexamethylene (CH₂)₂ Biguanide (CH₂)₆ Biguanide biguanide(PEHMB) Polypropylene biguanide, (CH₂)₃ Biguanide (CH₂)₃ BiguanidePolyaminopropyl biguanide (PAPB) Poly-[2-(2-ethoxy)-ethoxyethyl]-(CH₂CH₂O Biguanide (CH₂CH₂OCH₂ Biguanide biguanide-chloride] CH₂CH₂OCH₂OCH₂CH₂) (PEEG) CH₂CH₂) Polypropylenehexamethylene (CH₂)₃ Biguanide(CH₂)₆ Biguanide biguanide Polyethyleneoctamethylene biguanide (CH₂)₂Biguanide (CH₂)₈ Biguanide Polyethylenedecamethylene biguanide (CH₂)₂Biguanide (CH₂)₁₀ Biguanide Polyethylenedodecamethylene (CH₂)₂ Biguanide(CH₂)₁₂ Biguanide biguanide Polytetramethylenehexamethylene (CH₂)₄Biguanide (CH₂)₆ Biguanide biguanide Polytetramethylenebiguanide (CH₂)₄Biguanide (CH₂)₄ Biguanide Polypropyleneoctamethylene (CH₂)₃ Biguanide(CH₂)₈ Biguanide biguanide Polytetramethyleneoctamethylene (CH₂)₄Biguanide (CH₂)₈ Biguanide Biguanide Polyhexamethylene (CH₂)₆ BiguanideCH₂—CH₂—NH— Biguanide diethylenetriamine biguanide CH₂—CH₂Polyhexamethylene guanide (CH₂)₆ guanidine (CH₂)₆ guanidine (PHMG)Polyethylene guanide (CH₂)₂ guanidine (CH₂)₂ guanidinePolyethylenetetramethylene guanide (CH₂)₂ guanidine (CH₂)₄ guanidinePolyethylene hexamethylene (CH₂)₂ guanidine (CH₂)₆ guanidine guanidePolypropylene guanide, (CH₂)₃ guanidine (CH₂)₃ guanidine Polyaminopropylguanide (PAPB) Poly-[2-(2-ethoxy)-ethoxyethyl]- (CH₂CH₂O guanidine(CH₂CH₂OCH₂ guanidine guanide CH₂CH₂O CH₂OCH₂CH₂) CH₂CH₂)Polypropylenehexamethylene guanide (CH₂)₃ guanidine (CH₂)₆ guanidinePolyethyleneoctamethylene guanide (CH₂)₂ guanidine (CH₂)₈ guanidinePolyethylenedecamethylene guanide (CH₂)₂ guanidine (CH₂)₁₀ guanidinePolyethylenedodecamethylene (CH₂)₂ guanidine (CH₂)₁₂ guanidine guanidePolytetramethylenehexamethylene (CH₂)₄ guanidine (CH₂)₆ guanidineguanide Polypropyleneoctamethylene guanide (CH₂)₃ guanidine (CH₂)₈guanidine Polytetramethylene guanide (CH₂)₄ guanidine (CH₂)₄ guanidinePolyhexamethylene (CH₂)₆ guanidine CH₂—CH₂—NH— guanidinediethylenetriamine guanide CH₂—CH₂CAS Numbers for Example Compounds Arising from Formula 1a

Polymer CAS Number Polyhexamethylene biguanide hydrochloride (PHMB)27083-27-8 32289-58-0 Polyhexamethylene guanidine hydrochloride (PHMG)57028-96-3 Poly-[2-(2-ethoxy)-ethoxyethyl]-guanidinium-chloride]374572-91-5 (PEEG)

TABLE 2 Examples of polymer analogues arising from formula 1b. Name L₃G₄ L₄ G₅ x Polyallylbiguanide (CH₂—CH) Biguanide (CH₂—CH) Biguanide CH₂poly(allylbiguanidnio-co- (CH₂—CH) amine (CH₂—CH) biguanide CH₂allylamine) poly(allylcarbamoylguanidino- (CH₂—CH) amine (CH₂—CH)Carbamoyl CH₂ co-allylamine) guanidine polyvinylbiguanide (CH₂—CH)Biguanide (CH₂—CH) biguanide absent

The entry-promoting agent used in the method of the invention maycomprise linear, branched or dendrimeric molecules. The entry promotingagent may comprise a combination of linear, branched or dendrimericmolecules. The entry promoting agent may comprise one or any combinationof molecules of Formula 1a or formula 1b, for example as describedabove.

For example, the entry-promoting agent can comprise one or more ofpolyhexamethylene biguanide (PHMB), polyhexamethylene monoguanide(PHMG), polyethylene biguanide (PEB), polytetramethylene biguanide(PTMB) or polyethylene hexamethylene biguanide (PEHMB). Some examplesare listed in table 1 and 2.

Thus, the entry-promoting agent may comprise homogeneous orheterogeneous mixtures of one or more of polyhexamethylene biguanide(PHMB), polyhexamethylene monoguanide (PHMG), polyethylene biguanide(PEB), polytetramethylene biguanide (PTMB), polyethylene hexamethylenebiguanide (PEHMB), polymethylene biguanides (PMB),poly(allylbiguanidnio-co-allylamine), poly(N-vinylbiguanide),polyallybiguanide

The compounds can be synthesised in the laboratory by standardprocedures or may be obtained from commercial suppliers, as will be wellknown to those skilled in the art.

PHMB, for example, may also have synonyms poly(hexamethylene)biguanidehydrochloride; polymeric biguanide hydrochloride; polyhexanide;biguanide; CAS Number 27083-27-8; 32289-58-0; IUPAC namePoly(iminoimidocarbonyl)iminohexamethylene hydrochloride. PHMB can besynthesised in the laboratory by standard procedures or may be obtainedfrom suppliers, for example, Arch(http://www.archchemicals.com/Fed/BIO/Products/phmb.htm). Typically n=2to 40, average n:11, average molecular weight: 3025. PHMB is sold as abiocide, for example for use in hygiene products, swimming pool watertreatment and wound dressings.

Polyhexamethylene monoguanide (PHMG) can be synthesised in thelaboratory by standard procedures or obtained from suppliers, forexample from Shanghai Scunder Industry Co., Ltd,http://scunderen.busytrade.com/products/info/683633/PHMG.html

As will be appreciated by those skilled in the art, the entry-promotingpolymer may be a copolymer or heteropolymer ie the monomers may not beintended to be identical. However, typically the monomer units may beintended to be identical.

The entry promoting polymer in the present invention can be used fordelivery into both prokaryotic and eukaryotic cells. Thus, the cell maybe a prokaryotic cell. Examples of such cells will be well known tothose skilled in the art and include Gram negative bacteria; Grampositive bacteria; and mycobacteria or acid fast bacteria, for exampleMycobacterium smegmatis. Examples of Gram-negative bacteria include E.coli, S. enterica, for example S. entericia serovar Typhimurium,Salmonella spp and Campylobacter spp. Examples of Gram-positive bacteriainclude S. aureus.

The cell may alternatively be a eukaryotic cell, for example a fungalcell, for example Aspergillus, for example A. fumigatus; Candida spp;Saccharomyces spp; Pichia spp. The cell may be a mammalian cell (whichmay be a cell in cell culture, or a cell present in a tissue or organ).The cell may, for example, be a human, mouse, rat, rabbit, bovine or dog(or, for example, any other wild, livestock/domesticated animal) cell.The cell may, for example, be a stable cell line cell, or a primarycell, adherent or suspension cell. As examples, the cell may be amacrophage, osteosarcoma or HeLa cell line cell or a mouse primary cell.

The eukaryotic cell may alternatively be a plant cell (for example amonocotyledonous or dicotyledenous plant cell; typically anexperimental, crop and/or ornamental plant cell, for exampleArabidopsis, maize); fish (for example Zebra fish; salmon), bird (forexample chicken or other domesticated bird), insect (for exampleDrosophila; bees), Nematoidia or Protista (for example Plasmodium spp orAcantamoeba spp) cell.

The introduced agent may typically be or comprise a bioactive compound,for example a pharmaceutically active substance, or a diagnostic/imagingtool or probe, typically that has poor cell uptake properties. Theintroduced agent may comprise a nucleic acid or nucleic acid analogue.The nucleic acid or nucleic acid analogue may, for example, be orcomprise DNA or RNA or both. The nucleic acid or nucleic acid analoguemay typically be an antisense nucleobase oligomer, or a sense nucleobaseoligomer (for example encoding a polypeptide or a structural orregulatory nucleic acid). The nucleobase oligomer may be any type ofnucleic acid analogue or mimic that retains the capacity for basepairing, as will be well known to those skilled in the art. For example,the nucleic acid/analogue or nucleobase oligomer may be aphosphorothioate, 2′O-methyl nucleic acid, locked nucleic acid, peptidenucleic acid (PNA) oligomer. Alternatively, the nucleic acid/analogue ornucleobase oligomer may be, for example, a phosphorothioate, morpholinooligomer (PMO). The skilled person will readily be able to determinewhether a given nucleobase oligomer chemistry is compatible with thenucleobase oligomer retaining the ability to bind specifically to atarget nucleic acid, for example to act as a probe, guidepolypeptide/nucleic acid expression, or mediate gene silencing (forexample through antisense or RNAi, as will be well known to thoseskilled in the art). The skilled person will also readily be able todetermine whether a given nucleobase oligomer chemistry is compatiblewith promotion of entry by the entry-promoting agent: it is consideredthat an entry-promoting agent as set out above is useful generally withnucleic acids/analogues/nucleobase oligomers.

PNA and morpholino oligomers typically have uncharged (rather thananionic) backbones. There has previously been very modest success in PNAdelivery. Many strategies that work with DNA and RNA do not work withPNA and morpholino oligomers, which are typically uncharged. We havesurprisingly found that the entry-promoting agent of the presentinvention works with such molecules.

The nucleic acid/analogue/nucleobase oligomer may be single stranded ordouble stranded. The nucleobase oligomer may be able to act as a probe,in guiding polypeptide/nucleic acid expression, or as an RNAi molecule,for example small interfering RNA (siRNA) or microRNA, as an aptamer orligand as is well known to those skilled in the art. Typically thenucleic acid/analogue/nucleobase oligomer may be single stranded,particularly when the cell is prokaryotic. When the cell is eukaryotic(for example when the cell is a yeast or other fungus, or is a mammaliancell), the nucleobase oligomer may typically be single stranded ordouble stranded, which may include a mixture of single and doublestranded regions.

The nucleobase oligomer can be any molecule that hybridizes by asequence specific base pairing to a complementary DNA and/or RNAsequence. In the context of this invention, “hybridization” meanshydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases. For example, adenine and thymine are complementarynucleobases which pair through the formation of hydrogen bonds.

Further relevant features of nucleobase oligomers, for example antisensenucleobase oligomers, will be well known to those skilled in the art,and are, for example, set out in WO 02/079467 (hereby incorporated byreference), for example on page 2, lines 1 to 34 (antisense RNAregulation; PNA preparation, use as an antisense compound; cell uptake);page 6, lines 14 to 34 (hybridization to target sequence); page 8, lines1 to 25 (types of nucleobase oligomer); page 8, line 27 to page 14, line2 (length of antisense compounds; types of more on types of nucleobaseoligomer); page 14, line 19 to page 15, line 19 (cell uptake; lengthconsiderations); page 15, line 29 to page 16, line 26 (cell uptake);page 16, line 28 to page 17, line 6 (synthesis); page 18, line 33 topage 19, line 6 (linker connection between PNA and peptide).

The nucleobase oligomer may comprise, for example, phosphorothioate,2′O-methyl, 2′Fluoro, locked nucleic acid (LNA), morpholino, PNA ordeoxy nucleotides.

See, for example:

Phosphorothioate

http://www.ncbi.nlm.nih.gov/pubmed/1772569

LNA

http://www.pnas.org/content/97/10/5633.short

PNA

http://www.ncbi.nlm.nih.gov/pubmed?term=Progress%20in%20Developing%20PNA%20as%20a%20Gene-Targeted%20Drug

morpholino

http://www.liebertonline.com/doi/abs/10.1089/oli.1.1997.7.187

The introduced agent may comprise plasmid or other vector DNA (which maybe modified DNA), as will be well known to those skilled in the art.Typically plasmid or vector DNA may encode and/or be suitable forexpressing (or promoting expression of, for example by encoding acellular factor which, when expressed, activates the expression of anendogenous gene) a polypeptide or nucleic acid of interest.Alternatively, the introduced agent may comprise an RNA (which may bemodified RNA, for example as discussed above) molecule, for example theintroduced agent may comprise an siRNA molecule ie a molecule capable ofmediating RNA interference, as well known to those skilled in the art.

Successfully transformed or transfected cells, ie. cells that contain aDNA construct as noted above, can be identified by well knowntechniques. For example, one selection technique involves incorporatinginto the expression vector a DNA sequence (marker) that codes for aselectable trait in the transformed cell. These markers includedihydrofolate reductase, G418 or neomycin resistance for eukaryotic cellculture, and tetracycline, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria. Alternatively, the gene forsuch selectable trait can be on another vector, which is used toco-transform the desired host cell.

The marker gene can be used to identify transformants but it isdesirable to determine which of the cells contain recombinant DNAmolecules and which contain self-ligated vector molecules. This can beachieved by using a cloning vector where insertion of a DNA fragmentdestroys the integrity of one of the genes present on the molecule.Recombinants can therefore be identified because of loss of function ofthat gene.

Successfully transformed cells, ie cells that contain a DNA construct asnoted above, can be identified by well known techniques. For example,another method of identifying successfully transformed cells involvesgrowing the cells resulting from the introduction of an expressionconstruct as noted above can be grown to produce the encodedpolypeptide. Cells can be harvested and lysed and their DNA contentexamined for the presence of the DNA using a method such as thatdescribed by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al(1985) Biotech. 3, 208. Alternatively, the presence of the protein inthe supernatant can be detected using antibodies as well known to thoseskilled in the art.

In addition to directly assaying for the presence of recombinant DNA,successful transformation can be confirmed by well known phenotypicassays, for example immunological methods, when the recombinant DNA iscapable of directing the expression of the protein. For example, cellssuccessfully transformed or transfected with an expression vectorproduce proteins displaying appropriate antigenicity. Samples of cellssuspected of being transformed or transfected are harvested and assayedfor the protein using suitable antibodies.

Methods for determining whether an antisense reagent has been taken upby a cell will also be well known to those skilled in the art, forexample methods similar in concept to those described above; theintroduced antisense agent may result in a reduction in endogenous genefunction or expression.

The introduced agent may comprise a polypeptide (by which term isincluded smaller polypeptides or fragments of larger polypeptides, whichmay be termed peptides, for example of fewer than 50 amino acids inlength, typically 10-20 amino acid residues in length; as well as largeror full length polypeptides which may be termed proteins, for example ofmore than 50 amino acids in length).http://en.wikipedia.org/wiki/Peptide. The term polypeptide is intendedto encompass peptidomimetic compounds, as will be well known to thoseskilled in the art. One example of peptidomimetic compounds is discussedin Sherman and Spatola, J. Am. Chem. Soc., 112: 433 (1990). Thepolypeptide may be a therapeutic polypeptide, for example a polypeptidethat replaces the function of a missing/depleted and/or defectivepolypeptide; or may be an antigenic polypeptide, for example apolypeptide intended to serve as a vaccine, or may act as a ligand thatbinds to another protein or peptide. For example, the polypeptide may bean agonist or antagonist of a cellular protein. The delivered peptide orprotein may act by either inhibiting or promoting an enzyme's functionin a cell to build understanding of the role of the enzyme in a cell orto have a therapeutic or other useful effect in the cell, or in thetissue, organ or body of which the cell may be a part.

Methods for determining whether a polypeptide has been taken up by acell will also be well known to those skilled in the art. For examplethe protein may have fluorescence properties, such as green fluorescenceprotein (GFP) that can be observed and monitored using fluorescencemicroscopy, flow cytometry or similar methods. Also, uptake may beassessed using antibody-mediated staining. Finally, diverse functionalstudies can be utilized, according to the known function of the protein.For example, if the protein is a transcription factor, genes regulatedby the factor could be profiled to assess changes in expression levels.Alternatively, the protein may be a kinase, and changes inphosphorylation of its known substrates could be measured usingphosphorylation assays. Methods for determining whether a peptide hasbeen taken up by a cell will also be well known to those skilled in theart. For example the peptide may be fluorescently labelled, using forexample fluorescein, which can be observed and monitored usingfluorescence microscopy, flow cytometry or similar methods. Also, uptakemay be assessed using antibody-mediated staining. Finally, diversefunctional studies can be utilized, according to the known function(s)of proteins and peptides. Alternatively, if the peptide is a ligand, thedownstream effects of peptide interaction with a receptor could beassessed. If the protein/peptide is a toxin, the toxic effects could beassayed. Finally many peptides and proteins are immunogenic and theimmune function effects following delivery can be assessed using methodsthat are well known to molecular and cellular immunologists and othersskilled in the art.

The introduced agent may comprise a small drug or bioactive reagent, forexample having a molecular weight of less than 1000 or 2000 Da. Thesmall drug or bioactive reagent may have poor cell uptake or stabilityproperties in the absence of the entry-promoting agent of the presentinvention. An example is gentamycin, which is a useful antibiotic, butdoes not readily enter host cells. A second example is amphotericin B.Negatively charged or highly hydrophobic molecules may be introducedagents that may benefit from the delivery technology of the presentinvention.

Methods for determining whether a small molecule has been taken up by acell will also be well known to those skilled in the art. The smallmolecule may have inherent fluorescent properties allowing uptake to bemonitored using the methods described about. Also, radioisotopes can beincorporated, or small fluorophores may be conjugated providingfluorescent properties. For example a DNA ligand small molecule thatincreases in fluorescence upon binding to DNA provides a convenientassay for delivery, showing both cellular uptake and binding to thetarget molecule. In many cases small molecule delivery may be assessedthrough functional analyses of effects caused by binding to itsreceptor. For example, the small molecule may be an agonist or anantagonist and downstream effects can be measured using methods that arewell known to those skilled in the art.

The introduced agent may comprise a cellular imaging probe, for examplea contrast agent for in vivo imaging such as quantum dots,superparamagnetic iron oxide; or a receptor ligand, for example for anintracellular receptor, for example a nuclear hormone receptor; or anucleic acid based imaging probe, as noted above, all of which will bewell known to those skilled in the art. Methods used to determine thelocation of imaging probes are well known to those skilled in the artand include, but are not limited to, fluorescence imaging andradioactivity monitoring.

The introduced agent and the entry-promoting agent may be covalentlyjoined. Alternatively, the introduced agent and the entry-promotingagent may be provided as a formulation, for example as a non-covalentcomplex. The formulation may be prepared by mixing the entry-promotingagent and the introduced agent in appropriate ratios and underappropriate conditions of, for example, pH and salt concentration, forexample as set out in the examples. The method may, for example, beperformed from up to 100 fold molar excess of introduced agent overentry-promoting agent, through using an equal molar concentration ofcarrier and cargo molecules, to up to 1000 fold molar excess ofentry-promoting agent over introduced agent. For example, an appropriatemolar ratio of introduced agent (for example nucleic acid, for exampleoligonucleotide, for example of 10-30 bases in length andentry-promoting agent may be in the range of 1:0.1 to 1:50 or 1:0.5 to1:1000, for example 1:1 to 1:10 or 1:5, for example around 1:1.5. Anappropriate weight:weight ratio of introduced agent and entry-promotingagent may be in the range of 1:0.1 to 1:50 or 1:0.5 to 1:1000, forexample 1:1 to 1:10 or 1:5, for example around 1:1.5. The formation ofcomplexes is discussed further below. The pH at which theentry-promoting agent and the introduced agent are mixed/incubated maybe a high pH, for example 10-13.5, as discussed further below.

The method of the first aspect of the invention may be performed invitro. Examples of situations in which the method of the invention maybe useful include large-scale batch transfections and experimentaltransfections, for example for expressing a valuable protein or tounderstand the role of a gene or conditions that affect the gene or geneproduct. Also, the method may be used to characterise a microbiologicalsample. As noted above, the invention may be useful in, for example,functional studies; generation of stable cell lines; gene silencing; DNAvaccination; and drug delivery.

Thus, a further aspect of the invention provides a method for making atarget polypeptide (which term, as noted above, includes both peptidesand proteins, including covalently modified polypeptides, for exampleglycosylated polypeptides, as appropriate), the method comprising thestep of preparing the target polypeptide from a cell culture of a hostcell, wherein the host cell is a host cell that has been transformed (orwhose progenitor has been transformed) by an exogenous nucleic acidmolecule (which may be a copy of an endogenous nucleic acid molecule,but which typically is a nucleic acid molecule that differs from nucleicacid molecules previously present within the cell) so that the cellsynthesises the target polypeptide, wherein the transfection comprisesexposing the host cell to the exogenous nucleic acid molecule in thepresence of an entry-promoting agent as defined in relation to the firstaspect of the invention.

The method may comprise the step of culturing a host cell underconditions for synthesising the target polypeptide, as will be wellknown to those skilled in the art. Typically the method may be performedin vitro but it will be appreciated that the method may also beperformed in vivo, for example in a plant or (non-human) animal.

The selection of appropriate host cell and other factors such as cultureconditions may readily be performed by the skilled person for theintended target polypeptide.

The method of the first aspect of the invention may be performed in vivoor ex vivo. For example the method may be performed on a body surface,for example skin or mucosal membrane. The method may be performed oncells that are subsequently introduced or returned to a subjectorganism, for example mammal, for example human orlivestock/companion/laboratory animal.

A further aspect of the invention provides an entry-promoting agent asdefined in relation to the first aspect of the invention and anintroduced agent as defined in relation to the first aspect of theinvention for use in treating a subject in need of the introduced agent.

Similarly, a further aspect of the invention provides the use of anentry-promoting agent as defined in relation to the first aspect of theinvention and an introduced agent as defined in relation to the firstaspect of the invention in the manufacture of a medicament for use intreating a subject in need of the introduced agent.

A further aspect of the invention provides a method of treating asubject in need of an introduced agent as defined in relation to thefirst aspect of the invention, the method comprising the step oftreating the subject with an entry-promoting agent as defined inrelation to the first aspect of the invention and the introduced agent.

The subject may be, for example, a mammal, for example a human or alivestock/companion/laboratory/wild animal.

The skilled person will readily appreciate that the present inventionmay be useful in relation to a wide range of disease or conditions, forexample diseases or conditions in which treatment with siRNA reagents ortherapeutic or antigenic polypeptides may be useful. As examples, theinvention may be useful in relation to a mouthwash for treating oralviral infections, for example the common cold, for example with theentry-promoting agent and a nucleic acid against the virus strain inquestion; for treatment of skeletal related diseases, where theentry-promoting agent is combined with polypeptide or nucleic acid andinjected into a joint as an emulsion or suspension to treat arthritis;delivery (for example to the skin) of a DNA vaccine (ie DNA encoding anantigenic polypeptide or vaccine against cancer or other disease e.g.analogous to DNA electroporation vaccines into skin (targeting the DC's)but as a topical agent instead; treatment (for example topicaltreatment) of a skin disease (for example acne, psoriasis) for example anucleic acid based therapeutic. However, many other uses of theinvention are envisaged, for example corresponding to the range ofsituations in which RNAi, DNA or polypeptide therapeutics are consideredto be useful, particularly with delivery to a body surface, as notedabove.

A further aspect of the invention provides a kit of parts or compositioncomprising an entry-promoting agent and an introduced agent as definedin relation to the first aspect of the invention. The kit of parts maybe intended to, or composition may, comprise between a 100 fold molarexcess of introduced agent over entry-promoting agent, through an equalmolar concentration of carrier and cargo molecules, up to 1000 foldmolar excess of entry-promoting agent over introduced agent. Forexample, an appropriate molar ratio of introduced agent (for examplenucleic acid, for example oligonucleotide, for example of 10-30 bases inlength and entry-promoting agent may be in the range of 1:0.1 to 1:50 or1:0.5 to 1:1000, for example 1:1 to 1:10 or 1:5, for example around1:1.5. An appropriate weight:weight ratio of introduced agent andentry-promoting agent may be in the range of 1:0.1 to 1:50 or 1:0.5 to1:1000, for example 1:1 to 1:10 or 1:5, for example around 1:1.5.Preferences for the entry-promoting agent and introduced agent are asset out above. For example, the introduced agent may be siRNA molecules.The formation of complexes is discussed further below. The pH at whichthe entry-promoting agent and the introduced agent are mixed/incubatedmay be a high pH, for example 10-13.5, as discussed further below.

A further aspect of the invention provides a kit of parts or compositionof the invention wherein the kit of parts or composition ispharmaceutically acceptable. Thus, the kit components or composition maycomprise (or consist of) pharmaceutically acceptable components, as willbe well known to those skilled in the art. Entry-promoting agents of thepresent invention are considered to be pharmaceutically acceptable.PHMB, for example, is already used in, for example, wound dressings.

A further aspect of the invention provides a composition or kit of partsof the invention for use in treating a patient in need of the introducedagent.

A further aspect of the invention provides a composition or kit of partsof the invention for use in an imaging or diagnostic method. Forexample, as discussed above, the introduced agent may be an agent usefulin imaging or otherwise detecting an intracellular component, forexample a nucleic acid or protein. Thus, for example, the introducedagent may be an agent that binds specifically to an intracellularcomponent. For example, the introduced agent may be an antibody orantibody fragment typically retaining specific binding affinity for aparticular antigen, as well known to those skilled in the art; or anucleic acid that hybridises with the required degree of specificity toa particular nucleic acid sequence. As will be appreciated, the imagingor diagnostic method is typically a medical imaging or diagnostic methodand may typically be performed on the subject or may be performed on asample obtained from a subject or may be performed on some other sample.For example, the sample may be a food or water sample or otherenvironmental sample, as will be well known to those skilled in the art.

Thus, for example, an entry-promoting agent of the invention and anintroduced agent as defined above, for example a nucleic acid/analogueor nucleobase oligomer, may be used in the detection of an intracellularmolecule, for example in the detection of a polypeptide or a nucleicacid sequence, for example by in-situ hybridisation, for example wherethe delivered nucleic acid is labelled with a fluorophore or otherdetectable molecule.

The kit of parts or composition of the invention may further comprise acell for receiving the introduced agent. The cell may be, for example, acell useful in expressing a polypeptide whose expression is encoded orinduced by the introduced agent.

In an embodiment, the entry promoting agent and delivered molecule maybe provided together in a buffer having a high pH. Thus, the method forpromoting entry of an agent (introduced agent) into a cell may comprisethe step of exposing the cell to the introduced agent in the presence ofan entry promoting agent (all as set out above) wherein the introducedagent and the entry promoting agent have been mixed or incubated at highpH, for example in a buffer having high pH. The term “high pH” will bewell known to the skilled person and typically indicates a pH of above9, for example above 9.5 or above 10, for example between 10 and 13.5.Typically the introduced agent and the entry promoting agent are mixedat high pH to form nanoparticles, before exposing the cell to theintroduced agent in the presence of the entry promoting agent.

Specifically, buffers (with or without added salts, for example ascommonly used in molecular biology buffers, for example PBS; NaCl; ormany others) in the range of pH 10-13.5 are considered to provideformulations with improved transfection efficiencies (as shown in FIG.34). Resulting complex can be diluted 1:1 to 1:1000 in a suitable growthmedium, even complex can be added at several time points to cells(repeated multiple transfection) to achieve more efficiency. Theprocedure involves separate dilution of both the entry promoting agentand the delivered molecule in buffers with high pH and mixing them toform nanoparticles. Ratios and concentrations of the entry promotingagent and the introduced agent may be as discussed above in relation topreparation of a formulation and non-covalent complex; and in relationto the kit of parts.

For Example, the Following Procedure can be Used:

Dilute 1-100 μg of the entry promoting agent in buffer, for example 50μl. Dilute 0.01-50 μg of plasmid DNA in for example 50 μl of buffer. Mixthese two solutions and incubate, for example at room temperature for 1minute to several hours. This mixture can then be used in transfectionreactions, using well know methods. Add an appropriate volume of growthmedium to the entry promoting agent/delivered molecule mixture, mix andadd to cells growing in culture. Transfection will occur as the cellculture is incubated under appropriate conditions know to those workingwith cell culture.

Those skilled in the art will appreciate that high pH buffers can beeasily prepared using, for example, NaOH or KOH. These buffer conditionsprovide improve transfection efficiencies when using typicalcomplexation times, for example 30 minutes. Therefore, high pH buffers(and the entry promoting agents set out herein) can be easilyincorporated into the protocols currently used by researchers.

A further aspect of the invention provides a method for preparing acomplex comprising an entry promoting agent (for example PHMB) and anintroduced agent as defined above, the method comprising incubating theentry promoting agent and the introduced agent in a complexation buffer,for example at a high pH, for example at a pH of 10-13.5. It isconsidered that nanoparticles are formed comprising the entry promotingagent (for example PHMB) and the introduced agent, for exampleoligonucleotide polymers (DNA, PNA, siRNA), proteins, peptides and smallmolecules. Specifically, formation of nanoparticles can be achieved byincubating PHMB and similar molecules as described above witholigonucleotides, proteins, peptides and small molecules in anappropriate buffer prior to use with cells. An appropriate incubationbuffer may include water, PBS, and other buffers used commonly inlaboratories. High pH buffers are described above. The optimal buffermay depend on the specific identity of both the entry promoting agentand the delivered molecule, as will be apparent to those skilled in theart. Nanoparticle formation and cell delivery typically is achieved bydilution of both partner molecules in complexation buffer prior tomixing the two components. Also, mixing of the two components typicallyis carried out prior to combination with other excipients or activeingredients and application to cells or use in vivo. Efficientnanoparticle formation is considered to occur within seconds or minutesbut the procedure may be carried out over a number of hours. Anappropriate ratio for efficient nanoparticle formation varies withdifferent partner combinations. For example, 1-20:1 (wt:wt) forPHMB:plasmid DNA provides efficient nanoparticle formation. Examples aregiven above for PHMB;DNA combinations that result in nanoparticleformation. A person skilled in the art will be able to assessnanoparticle formation and delivery efficiencies when using differentpartner molecule ratios. Nanoparticle formation can be assessed in anumber of ways. For example, an individual skilled in the art will beable to assess nanoparticle formation using dynamic light scattering(DLS) and microscopy methods.

A further aspect of the invention provides a complex comprising an entrypromoting agent (for example PHMB) and an introduced agent as definedabove, wherein the complex is obtainable (or obtained) by a methodcomprising incubating the entry promoting agent and the introduced agentin a complexation buffer, for example at a high pH, for example at a pHof 10-13.5.

The complexation buffer may in each case alternatively or in additioncomprise a cross-linking agent, for example as set out in FIG. 40, forexample 1,4-butanediol glycidylether or similar.

The invention is now described in more detail by reference to thefollowing, non-limiting, Figures and Examples.

FIG. 1 PHMB and possible mechanism of action of PHMB as an antibacterialagent

FIG. 2 Conjugation of PHMB with FITC

FIG. 3 Uptake of free PHMB into bacterial cells

FIG. 4 PHMB enters into wide variety of bacterial cells.

Uptake of PHMB into Gram negative and acid fast bacteria: overnightcultures were treated with 1 μM PHMB-FITC, for 1 hr and counter stainedwith DAPI before observing under fluorescence microscope. PHMB hasentered into all the cells as shown by the presence of green cells underFITC filter.

FIG. 5 PHMB-FITC enters into Gram positive bacteria

Uptake of PHMB inot Gram positive bacterial cells: Overnight cultures ofS. aureus wild type were treated with 1 μM PHMB-FITC, for 1 hr andcounter stained with DAPI. All the cells in a given field are positivefor PHMB uptake, indicating the efficient cell penetration.

FIG. 6 PHMB enters efficiently into fungi

Aspergillus fumigatus vegetative cells were treated with PHMB-FITC. Itis apparent that the PHMB enters into all the cells.

FIG. 7 Phase contrast image of PHMB treated Aspergillus fumigatus

Phase contrast images showing the fungal hyphae and entry of PHMB intomost of the hyphae. Fungal cells are relatively difficult to transfect.Uptake of PHMB into Aspergillus hyphae show the potential utility ofthis molecule as a carrier for fungi.

FIG. 8 Entry of PHMB into Aspergillus fumigatus

FIG. 9 Uptake of free PHMB into mammalian cells

FIG. 10 PHMB enters into mammalian cells at a rapid rate

Uptake of free PHMB-FITC into J774 macrophages. Upper panel:fluorescence microscopy images of PHMB-FITC uptake. Lower panel:confocal images of time course measurements of PHMB uptake. PHMB startsentering the cells in less than 10 minutes.

FIG. 11 PHMB enters efficiently into adherent as well as suspensioncells

Left panel: uptake of PHMB into adherent cells (HEK 293). Right panel:uptake of PHMB into suspension cells (THP-1 monocytes).

FIG. 12 PHMB localizes to cytoplasmic compartment.

Cytoplasmic localization of PHMB in mammalian cells: HeLA cells treatedwith 10 μg/ml PHMB-FITC for 2 hr were observed under invertedfluorescence microscope, the arrow pointing the cytoplasmic localisationof the fluorescence. There is only background fluorescence in untreatedcells compared to PHMB treated cells, where all the cells are positivefor uptake.

FIG. 13 Interaction of PHMB with oligonucleotides

Evidence for the interaction of PHMB with nucleic acids: left panelshows the electrophoretic mobility of FAM labelled oligonucleotide. Lane1: Oligo oly, Lane 2-8: increasing PHMB/oligo molar ratio:: L2: 0.02,L3: 0.06, L4: 0.12, L5: 0.2, L6: 0.4, L7: 0.8, L8: 1.6. Addition of PHMBresults in the retardation of oligonucleotide migration, as evident inlane 5-7. At PHMB/oligo molar ratio 0.8, there is a complete retardationoin the migration, indicating an efficient nucleic acid binding. Rightpanel shows the spectroscopic evidence for the interaction of PHMB withFAM labelled oligonucleotide. Addition of PHMB results in quenching ofFAM fluorescence, which occurs when both molecules are in closeproximity, showing the interaction.

FIG. 14 Interaction of PHMB with plasmid DNA

Evidence for the interaction of PHMB with plasmid DNA: The complex ofPHMB/plasmid (encoding GFP) were prepared at different w/w ratio andincubated for 30 min at 37° C., then run on 1% agarose gel stained withethidium bromide (0.5 μg/ml). It is observed that addition of PHMBresults in retardation of migration of plasmid DNA 9 Lane 3-5). Completeretardation is observed at 2/2 ratio 1.5:1. This ratio was used as aguide for transfection experiments.

FIG. 15 Interaction of PHMB with siRNA

Evidence for the interaction of PHMB with siRNA: PHMB/siRNA complex wereprepared at different molar (M/M) ratio. It is observed that theaddition of PHMB resulted in retardation of migration of siRNA. At molarratio 20:1 complete retardation in migration is observed. This ratio wasused as a guideline for siRNA delivery.

FIG. 16 Interaction of PHMB analogues with nucleic acids

FIG. 17 Interaction of polyhexamethylene guanidine (PHMG) with nucleicacids

Gel shift assay to show the interaction of PHMG with nucleic acids.PHMG/plasmid (a) and PHMG/siRNA (b) complex were run on the agaraosegel. Complete retardation was observed for whole plasmid (w/w 2.5:1) andsiRNA (8:1, M/M).

FIG. 18 PHMB carries PNA into bacterial cells through noncovalentcomplex

Entry of PHMB/PNA-FITC complex into bacteria. E. coli AS19 and K12strains were treated with PHMB/PNA-FITC complex (molar ratio 11:1),counterstained with DAPI and observed under fluorescence microscope.There is a minimal uptake in PNA alone treated bacteria, as expected. Animprovement in the fluorescence intensity and number of positive cellswas apparent in cells treated with the PHMB/PNA complex.

FIG. 19 PHMB carries oligonucleotides into Aspergillus fumigatus

Uptake of PHMB/oligonucleotides into fungi. Aspergillus fumigatus wastreated with PHMB/FAM labelled oligo complex, counterstained with DAPI.There is a minimal uptake in oligonucleotide alone treated samples.Improvement in the uptake is noticed in PHMB/oligo complex treatedsamples, indicating the delivery of oligonucleotides into fungi.

FIG. 20 Delivery of oligonucleotides into mammalian cells

FIG. 21 PHMB carries oligonucleotides into nucleus of mammalian cells

Delivery of oligonucleotides into mammalian cells: HeLa cells weretreated with PHMB/oligo complex for 2 hours. There was no uptake incells treated with fee oligonucleotides, were as the cells treated withPHMB/oligonucleotide showed enhanced uptake. The oligonucleotides weredelivered into nucleus as indicated by the arrow.

FIG. 22 Delivery of plasmid encoding GFP into mammalian cells

FIG. 23 PHMB-delivered plasmid is able to express GFP in HeLa cells

Delivery of plasmid into mammalian cells. HeLa cells were treated withPHMB/plasmid encoding GFP. There was no expression of GFP in cellsuntreated (a) or treated with DNA alone (b, 500 ng plasmid). There wasan enhanced expression of GFP in PHMB/plasmid treated cells (c, 2.5 μgPHMB+500 ng plasmid). Lipofectamine 2000 was used as positive control(d).

FIG. 24 PHMB-delivered plasmid is able to express GFP in HeLacells

FIG. 25 PHMB delivered plasmid is able to express GFP in osteosarcomacells

Delivery of plasmid into mammalian cells. Osteosarcoma cells treatedwith PHMB/plasmid (c, 2.5:1, w/w) express GFP. There is no expression incells treated with plasmid alone (b) or untreated (a). Lipofectamine2000 was used as positive control (d).

FIG. 26 Uptake of PHMG-FITC into mammalian cells

Cell penetrating property of PHMG: HeLa cells were treated with 3 μg/mlPHMG-FITC (b). PHMG has entered efficiently into all the cells, whereasuntreated cells show a minimal background fluorescence (a). It isevident that analogues of PHMB also possess cell penetrating property.

FIG. 27 PHMG is also able to carry plasmid into mammalian cells

Delivery of plasmid into mammalian cells. Osteoscarma cells treated withPHMG/plasmid (a, 1.25:1) express GFP. This shows the carrier potentialof PHMG for mammalian cells. Lipofectamine 2000 is used as a positivecontrol (b).

FIG. 28 Delivery of siRNA into primary cells and analysing effects

FIG. 29 siRNA delivered by PHMB is able to silence genes in primarycells

Knockdown of Tbx gene in primary adipocytes. Tbx 15 siRNA pool was usedto knockdown Tbx 15 mRNA using lipofectamine and PHMB. 60% reduction inTbx 15 mRNA is observed when PHMB is used.

FIG. 30 PHMB is less toxic to primary cells than lipofectamine 2000

Cytotoxicity of carriers in primary cells. Cells were treated withlipofectamine 2000 and PHMB and observed 6 days post treatment. Bothlipofectamine and lipofectamine+siRNA treated cells show highercytotoxic effects than PHMB or PHMB/siRNA treated cells.

FIG. 31 PHMB forms nanoparticle with FITC

Characterization of particles formed by PHMB and cargo. Noncovalentinteraction between a carrier and cargo results in particle formation.PHMB/free FITC complex was prepared in water and the resulting size andsurface charge is analysed by dynamic light scattering. It is observedthat PHMB alone forms amorphous aggregate of mean size 888.6 nm, withPDI of (0.64) with surface charge of +42.8 mV indicating heterogeneouspopulation. PHMB/FITC complex had a mean size of 98.36 nm with PDI of0.20 indicating formation of homogenous population of nanoparticles,with positive surface charge (+12.2 mV).

FIG. 32 Analogues of PHMB as a potential transfection reagents

FIG. 33 Conjugation of PHMB with FITC, demonstrated by IR spectra

To a solution of 50 mg of PHMB and 50 μl N,N-diisopropylethylamine in800 μl deionized water, 2 mg of FITC in 100 μl DMF was added. Reactionmixture was shaken at room temperature overnight and then lyophilized,resulting in a residual mass, which was triturated with ethyl acetate toremove excess of unreacted FITC. The resulting material was dissolved in1 ml of deionized water and dialyzed (cut off 3.5 kDa) against 50%ethanol solution for 5 days with intermittent change of solution (10times, 500 ml). The dialyzed solution was lyophilized to obtainfluoresceinyl-PHMB. PHMB-FITC conjugation was confirmed by recording IRspectra, IR (Nujol), v (cm-1): 750-760 cm-1 (C═S stretching).

FIG. 34 High pH and salt enhances transfection using PHMB/pEGFPcomplexes

At left, effect of pH and buffer on complex formation and transfectionefficiency was tested by preparing buffers (water or PBS or 0.9% NaCl)with pH from 13.5-7 by using NaOH or HCl. Complex of 4 μg PHMB and 1 μgpEGFP was prepared in 100 μl volume of buffers by incubating at roomtemperature for 20 minutes and diluted in growth medium, added to1.5×105 HeLa cells at the time of plating in a 12 well plate.Transfection efficiency was monitored by Flowcytometry by measuring GFPexpression 36 hours post transfection.

At right, to indirectly assess the rate of complex formation betweenPHMB and pEGFP, multiple complex reactions were prepared by mixing 4 μgPHMB and 1 μg pEGFP in 1×PBS with pH 11.5, and added at different timepoints to HeLa cells in 12 well plate as describe above. GFP expressionwas measured by Flowcytometry 36 hours post transfection. PHMB formscomplexes with plasmid in less than 5 minutes and the reaction is stablefor several hours at room temperature.

FIG. 35 PHMB mediated delivery of a red fluorescent protein and antibody(IgG-Alexa488) into human cells (HeLa)

3 μg of PHMB was mixed with 0.3 μg of fluorescent proteinR-Phycoerythrin (panels at left) or alexa 488 labelled IgG antibody(panels at right) in 100 μl volume of PBS, allowed to stand for 30minutes at room temperature. The resulting complex was diluted in 900 μlgrowth medium and the diluted complexes were overlayed on HeLa cells.After 2 hours of incubation cells were imaged using a fluorescencemicroscope.

FIG. 36 PHMB mediated delivery of small fluorescent molecules into humancells (HeLa)

3.5 μg of PHMB was mixed with 1 μl of 100 μM small molecules (SYTOXGreen or FITC) in 100 μl of PBS, allowed to stand for 30 minutes at roomtemperature. The resulting complexes were diluted with 900 μl growthmedium and the diluted mixture was overlayed on HeLa cells, after 2hours of incubation cells were imaged under fluorescence microscope.

FIG. 37 PHMB forms nanoparticles with wide variety of cargo molecules

A. Characterization of nanoparticle formation by DLS.PHMB/oligonucleotides complex was prepared in 100 μl PBS by taking afixed amount of oligonucleotides (1 μl of 100 μM stock) and varying thePHMB concentration (1-6 μg), incubated for 30 minutes at roomtemperature, resulting complex was diluted in 1 ml filtered water andaverage size, zeta potential were monitored using Zetasizer ZS nano.PHMB/pEGFP complex was prepared in 100 μl PBS by taking a fixed plasmid(1 μg) and varying the PHMB concentration (1-6 μg), as described above.PHMB/siRNA complex was prepared in 100 μl PBS by taking a fixed siRNA (1μl of 100 μM stock) and varying the PHMB concentration (1-6 μg), asdescribed above. For calculating molar concentration, average molecularweight of PHMB was taken as 3000 Daltons.

B. Characterization of nanoparticle formation by fluorescencemicroscopy. PHMB/pEGFP complex were prepared as described above, plasmidwas stained with 100 nM SYBR Green and the complex were loaded on 1%agarose bed prepared on a glass slide and observed under fluorescencemicroscope using 490 nm Excitation, 520 nm emission. DNA alone doesn'tshow any particles, whereas PHMB/pEGFP complex appear as smallparticles.

FIG. 38 PHMB interacts with the small molecule Nystatin

At left, Interaction of PHMB with Nystatin (10 units/ml), indicated byquenching, monitored by recording emission at 710 nm, upon excitation at350 nm.

At right, Interaction of PHMB (3 mg) with Rifampicin (20 μg), indicatedby reduced absorbance, monitored by the quenching in absorbance (470 nm)in presence of PHMB over a period of time

FIG. 39 PHMB-mediated delivery of a fluorophore labelled DNAoligonucleotide into bacteria, Salmonella enterica

6 μg of PHMB was mixed with 1 μl of 100 μM 18 mer deoxyoligonucleotidelabelled with 6 FAMin 100 μl volume of water with Ph12, allowed to standfor 30 minutes at room temperature. The resulting complex was diluted in400 μl PBS, added to 100 μl of 0.2OD S. enterica in early log phase,mixed well and incubated at 37° C. for 2 hours and counter stained withDAPI and observed under fluorescence microscope. Oligonucleotidesdelivery was quantified by flowcytometry.

FIG. 40. Branched PHMB/PHMG mediated delivery of GFP expressing plasmidDNA into human cells

EXAMPLE 1 PHMB and Related Cationic Polymers as Carriers for Deliveryinto Bacteria, Fungi and Mammalian Cells

We have assessed PHMB and related molecules as carriers for deliveryinto prokaryotic and eukaryotic cells. No single technology has yetemerged as a practical gene transfer method for the clinic. Desirablequalities of a carrier include:

-   -   It should be able to interact with biomolecules    -   Enter on its own and as well as when loaded with biomolecules    -   Ability to release biomolecules upon entry    -   Low cytotoxic and suitable for in-vivo applications    -   High transfection efficiency for transient transfection is an        important feature for research, as are low cytotoxicity, and        effectiveness for difficult to transfect cells

We considered that PHMB and related molecules may be able to act aseffective carriers. Polyhexametheylenebiguanide (PHMB) is a cationicpolymer with broad spectrum antimicrobial agent and less toxic tomammalian cells (Vantocil™ Arch biocides limited, UK). Low toxicity isan unusual and very beneficial property among delivery reagents and thelow toxicity observed and safe track record of these compounds in abroad range of applications distinguishes this technology.

n=2 to 40, The weight-average molecular weight (Mw) and polydispersityfor Bx529 VANTOCIL 100 are 3035 and 1.9 respectively. Such a Mwcorresponds to an ‘n’ value of around 14 (more precisely 13.8). Endgroups: amine, guanidine and cyanoguanidine.Polyhexametheylenemonoguanide (PHMG) is an analogue of PHMB withantimicrobial properties (Zhou et al., 2010).

Current uses of PHMB:

-   -   Used as an antiseptic, recommended choice in surgical wound        dressings, (Stephen Gilliver2009)    -   present in disinfectants, swimming pool sanitizers, solid        surface cleanser, mouth rinses and contact lens solutions (Lucas        et al., 2009)    -   Used in treatment of hatching eggs, as a deodorizer and        preservative in cosmetics, textiles and treatment of cooling        systems to prevent Legionella growth etc

We considered that PHMB may be useful as a carrier, either as a covalentconjugate or as a non-covalent complex, possibly with hydrophobic and/orelectrostatic interactions. If this were to be the case, otherproperties of PHMB may enhance its use as a carrier. The toxicityprofile of PHMB is well studied (Muller et al., 2008; Kathryn V.Montague, 2004) and PHMB is considered to be a poor allergen (adesirable property) from tests in human patients, (Schnuch et al.,2007). The antimicrobial properties may also be advantageous in reducingthe problem of contamination in cell culture.

Uptake of free PHMB-FITC into three kingdoms was assessed:

a) Bacteria:

Gram negative: E. coli and S. enterica

Gram positive: S. aureus

Acid fast: M. smegmatis

b) Fungi: Aspergillus fumigatus

c) Mammalian cells: macrophages, monocytes, HEK and HeLa cells

See FIGS. 2 to 12, FIG. 33 for conjugation of PHMB with FITC

Evidence for interaction of PHMB with nucleic acids in-vitro (Essentialfor a carrier to form a noncovalent complex with cargo and deliver itinto cells)

See FIGS. 13 to 17.

Free PHMB enters into a wide variety of cells. PHMB interacts withnucleic acids. Can PHMB also carry nucleic acids and analogues? Yes: SeeFIGS. 18 to 21, FIG. 39.

Are the delivered nucleic acids available for effective function? Yes:see FIGS. 22 to 25

PHMB analogue also possess cell penetrating properties: see FIGS. 26 to27

Effect on primary cells: See FIGS. 28 to 30

Analogues: see FIGS. 31 to 32 and 40, (Note, the chemical formulae inFIG. 32 are adopted from F. C. Krebs et al./Biomedicine &Pharmacotherapy 59 (2005) 438-445).

EXAMPLE 2 Use of PHMB and Related Cationic Polymers for Small MoleculeDelivery into Mammalian Cells

We have shown that PHMB and related cationic polymers can interact withsmall molecules (for example Nystatin, Rifampicin, curcumin, free FITC,SYTOX Green) and deliver them into mammalian cells, for example HeLa.See FIGS. 31, 36 to 38. Also, we observed synergistic bacterial killingusing PHMB/rifampicin complexes that were formed prior to exposure tobacterial cells.

EXAMPLE 3 Use of PHMB and Related Cationic Polymers for Protein Deliveryinto Mammalian Cells

PHMB and analogues were able to deliver protein molecules (for exampleR-phycoerythrin or alexa 488 conjugated IgG antibodies) into mammaliancells such as HeLa cells, see FIG. 35.

EXAMPLE 4 PHMB Forms Complexes with Nucleic Acid Molecules Rapidly andHigh pH Enhances Transfection Efficiency

Our method shows that higher transfection efficiency of nucleic acidscan be achieved when carrier and cargo are complexed in buffers withhigh pH (13.5-10). We tested the amount of time required for complexformation between PHMB and nucleic acids, Our results show that aslittle as five minutes is sufficient to form complex which can entermammalian cells or even allowing the complex to stay for hours at roomtemperature will not compromise the transfection capacity. See FIG. 34.

EXAMPLE 5 Use of PHMB and Related Cationic Polymers to FormNanoparticles with Bioactive Molecules

We have assessed PHMB and related molecules as a means to formnanoparticles with a range of bioactive molecules. No single technologyhas yet emerged as a practical nanoparticle formation method for theclinic

Desirable qualities of a carrier include:

-   -   Efficient nanoparticle formation using simple procedures    -   Useful with a range of bioactive molecules    -   Ability to release biomolecules for bioactive effects    -   Low cytotoxic and suitable for in-vivo applications

We observed while investigating the cell delivery properties of PHMB andrelated molecules that these polymers are able to effectively formnanoparticles with a range of molecules, many of which are bioactive.The use of bioactive molecules may benefit from inclusion withinnanoparticles prior to further formulation and application. Suchbenefits could include improved solubility, improved stability,protection against degradation during use and improve or altereddistribution or clearance properties in vivo. Low toxicity is an unusualand very beneficial property among nanoparticle forming reagents and thelow toxicity observed and safe track record of these compounds in abroad range of applications over several decades of wide usagedistinguishes this technology.

Nanoparticle Formation with a Range of Bioactive Molecules was Assessed

Evidence for interaction of PHMB with nucleic acids in-vitro, which isessential for a carrier to form a noncovalent complex and nanoparticlewith cargo molecules and deliver it into cells. Evidence for complexformation was assessed using a range of methods

See FIGS. 13 to 17 and 38.

Dynamic light scattering (DLS) and fluorescence microscopy were used tomeasure the formation of nanoparticles. Evidence of nanoparticleformation was assessed using a range of molecules.

a) nucleic acids, including chromosomal DNA, plasmid DNA and RNA

b) proteins

c) small molecules

Our results show that PHMB and related molecules form nanoparticles withwide variety of cargo molecules. See FIGS. 31 and 37.

CONCLUSIONS

-   -   Free PHMB is able to enter a wide variety of bacteria, fungi and        mammalian cells (three kingdoms demonstrated)    -   PHMB and analogues are potent carriers for all cell types, with        large potential applications as a carrier for mammalian systems    -   PHMB and analogues interact with protein and peptides    -   PHMB interacts with a range of small organic molecules including        both basic and acidic small molecules.    -   PHMB and analogues are capable of carrying nucleic acids into        the nucleus of mammalian cells and presumably release them for        effective function inside cells    -   Very low toxicity is reported and observed for PHMB and        analogues    -   PHMB and analogues are capable of carrying proteins, peptides        and small molecules (example SytoxGreen and free FITC) into        mammalian cells.    -   PHMB and analogues are capable of carrying nucleic acids,        peptide nucleic acids and small molecules into bacteria and        fungi.    -   The substance cost is very low    -   Chemistry allows further modifications    -   Complexation of PHMB with nucleic acids under high pH results in        more efficient nucleic acid delivery into bacteria and mammalian        cells.    -   PHMB and analogues complex with nucleic acids very rapidly in        minutes and complex are stable for hours at room temperature    -   PHMB and analogues are able to form nanoparticles with a range        of bioactive molecules.

REFERENCES

-   Gilliver et al (2009) J Wound Care/ACtiva Healthcare Supplement 9-14    PHMB: a well-tolerated antiseptic with no reported toxic effects-   Müller & Kramer (2008) J Antimicrobial Chemotherapy 61, 1281-1287    Biocompatibility index of antiseptic agents by parallel assessment    of antimicrobial activity and cellular cytotoxicity-   Schnuch et al (2007) Contact Dermatitis 56, 235-239 The biocide    polyhexamethylene biguanide remains an uncommon contact allergen.-   Kathryn V. Montague (2004). Reregistration Eligibility Decision    (RED) for PHMB. United states environmental protection agency.    Washington, D.C. 20460.    (http://www.epa.gov/oppsrrd1/REDs/phmb_red.pdf)-   Lucas et al (2009) Talanta 80, 1016-1019. Analysis of    polyhexamethylene biguanide in multipurpose contact lens solutions.

1. A method for promoting entry of an agent (introduced agent) into acell, the method comprising the step of exposing the cell to theintroduced agent in the presence of an entry-promoting agent, whereinthe entry-promoting agent comprises a linear and/or branched or cyclicpolymonoguanide/polyguanidine, polybiguanide, analogue or derivativethereof according to the following Formula 1a or formula 1b:

wherein: “n”, refers to number of repeating units in the polymer, and ncan vary from 2 to 1000, for example from 2 or 5 to 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700,800 or 900; G₁ and G₂ independently represent a cationic groupcomprising biguanide or guanidine, wherein L₁ and L₂ are directly joinedto a Nitrogen atom of the guanide; L₁ and L₂ are linking groups betweenthe G₁ and G₂ cationic groups in the polymer and independently representan aliphatic group containing C₁-C₁₄₀ carbon atoms, for example an alkylgroup such as methylene, ethylene, propylene, C₄, C₅, C₆, C₇, C₈, C₉ orC₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀, -C₇₀, -C₈₀, -C₉₀, -C₁₀₀,-C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀, alkyl; or a C₁-C₁₄₀ (for example C₁, C₂,C₃, C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀,-C₇₀, -C₈₀, -C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀), cycloaliphatic,heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, or oxyalkyleneradical; or a polyalkylene radical optionally interrupted by one ormore, preferably one, oxygen, nitrogen or sulphur atoms, functionalgroups or saturated or unsaturated cyclic moiety; N and G₃ are optionalend groups; X can be either present or absent; L₃, L₄ and X are linkinggroups between the G₄ and G₅ cationic groups in the polymer andindependently represent an aliphatic group containing C₁-C₁₄₀ carbonatoms, for example an alkyl group such as methylene, ethylene,propylene, C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀-C₆₀, -C₇₀, -C₈₀, -C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀, alkyl; orL₃ and L₄ and X can independently be C₁-C₁₄₀ (for example C₁, C₂, C₃,C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀,-C₇₀, -C₈₀, -C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀), cycloaliphatic,heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, oxyalkyleneradicals; or a polyalkylene radical optionally interrupted by one ormore, preferably one, oxygen, nitrogen or sulphur atoms, functionalgroups as well as saturated or unsaturated cyclic moiety; “G₄” and “G₅”are cationic moieties and can be same or different, and at least one ofthem is a biguanidine moiety or carbamoylguanidine, and the other moietymay be biguanidine or carbamoylguanidine or amine; and cationic moietiesG₄ and G₅ do not contain single guanidine groups.
 2. The method of claim1 wherein the entry-promoting agent according to formula 1a comprises oris composed of one or more of the following: Name L₁ G₁ L₂ G₂Polyhexamethylene biguanide (CH₂)₆ Biguanide (CH₂)₆ Biguanide (PHMB)Polyethylene biguanide (PEB) (CH₂)₂ Biguanide (CH₂)₂ BiguanidePolyethylenetetramethylene (CH₂)₂ Biguanide (CH₂)₄ Biguanide biguanidePolyethylene hexamethylene (CH₂)₂ Biguanide (CH₂)₆ Biguanide biguanide(PEHMB) Polypropylene biguanide, (CH₂)₃ Biguanide (CH₂)₃ BiguanidePolyaminopropyl biguanide (PAPB) Poly-[2-(2-ethoxy)-ethoxyethyl]-(CH₂CH₂O Biguanide (CH₂CH₂OCH₂ Biguanide biguanide-chloride] CH₂CH₂OCH₂OCH₂CH₂) (PEEG) CH₂CH₂) Polypropylenehexamethylene (CH₂)₃ Biguanide(CH₂)₆ Biguanide biguanide Polyethyleneoctamethylene (CH₂)₂ Biguanide(CH₂)₈ Biguanide biguanide Polyethylenedecamethylene (CH₂)₂ Biguanide(CH₂)₁₀ Biguanide biguanide Polyethylenedodecamethylene (CH₂)₂ Biguanide(CH₂)₁₂ Biguanide biguanide Polytetramethylenehexamethylene (CH₂)₄Biguanide (CH₂)₆ Biguanide biguanide Polytetramethylenebiguanide (CH₂)₄Biguanide (CH₂)₄ Biguanide Polypropyleneoctamethylene (CH₂)₃ Biguanide(CH₂)₈ Biguanide biguanide Polytetramethyleneoctamethylene (CH₂)₄Biguanide (CH₂)₈ Biguanide Biguanide Polyhexamethylene (CH₂)₆ BiguanideCH₂—CH₂—NH— Biguanide diethylenetriamine biguanide CH₂—CH₂Polyhexamethylene guanide (CH₂)₆ guanidine (CH₂)₆ guanidine (PHMG)Polyethylene guanide (CH₂)₂ guanidine (CH₂)₂ guanidinePolyethylenetetramethylene guanide (CH₂)₂ guanidine (CH₂)₄ guanidinePolyethylene hexamethylene (CH₂)₂ guanidine (CH₂)₆ guanidine guanidePolypropylene guanide, (CH₂)₃ guanidine (CH₂)₃ guanidine Polyaminopropylguanide (PAPB) Poly-[2-(2-ethoxy)-ethoxyethyl]- (CH₂CH₂O guanidine(CH₂CH₂OCH₂ guanidine guanide CH₂CH₂O CH₂OCH₂CH₂) CH₂CH₂)Polypropylenehexamethylene (CH₂)₃ guanidine (CH₂)₆ guanidine guanidePolyethyleneoctamethylene guanide (CH₂)₂ guanidine (CH₂)₈ guanidinePolyethylenedecamethylene guanide (CH₂)₂ guanidine (CH₂)₁₀ guanidinePolyethylenedodecamethylene (CH₂)₂ guanidine (CH₂)₁₂ guanidine guanidePolytetramethylenehexamethylene (CH₂)₄ guanidine (CH₂)₆ guanidineguanide Polypropyleneoctamethylene (CH₂)₃ guanidine (CH₂)₈ guanidineguanide Polytetramethylene guanide (CH₂)₄ guanidine (CH₂)₄ guanidinePolyhexamethylene (CH₂)₆ guanidine CH₂—CH₂—NH— guanidinediethylenetriamine guanide CH₂—CH₂

or the entry promoting agent according to formula 1b comprises orconsists of poly(allylbiguanidnio-co-allylamine),poly(N-vinylbiguanide), poly(allylcarbamoylguanidno-co-allylamine) orpolyallybiguanide.
 3. The method of claim 1 wherein the cell is aprokaryotic cell.
 4. The method of claim 1 wherein the cell is aeukaryotic cell.
 5. The method of claim 1 wherein the cell is amammalian cell.
 6. The method of claim 1 where in the introduced agentcomprises a nucleic acid or nucleic acid analogue.
 7. The method ofclaim 6 wherein the introduced agent comprises plasmid DNA.
 8. Themethod of claim 6 wherein the introduced agent comprises an RNAimolecule.
 9. The method of claim 1 wherein the introduced agentcomprises a polypeptide.
 10. The method of claim 1 wherein theintroduced agent comprises a small drug, bioactive reagent or cellularimaging probe/contrast agent.
 11. The method of claim 1 where theintroduced agent and the entry-promoting agent are covalently joined.12. The method of claim 1 wherein the introduced agent and theentry-promoting agent are provided as a formulation, for example as anon-covalent complex, optionally as nanoparticles.
 13. The method ofclaim 1 wherein the method is performed in vitro.
 14. The method ofclaim 1 wherein the method is performed in vivo.
 15. The method of claim1 wherein the method is performed ex vivo.
 16. The method of claim 12wherein the method is performed with from up to 100-fold molar excess ofintroduced agent over entry-promoting agent to up to 1000-fold molarexcess of entry-promoting agent over introduced agent.
 17. A method formaking a target polypeptide, the method comprising the step of preparingthe target polypeptide from a cell culture of a host cell, wherein thehost cell is a host cell that has been transformed (or whose progenitorhas been transformed) by an exogenous nucleic acid molecule so that thecell synthesises the target polypeptide, wherein the transfectioncomprises exposing the host cell to the exogenous nucleic acid moleculein the presence of an entry-promoting agent as defined in any one of thepreceding claims.
 18. A composition comprising (i) an entry-promotingagent comprising a linear and/or branched or cyclicpolymonoguanide/polyguanidine, polybiguanide, analogue or derivativethereof according to the following Formula 1a or formula 1b:

wherein: “n”, refers to number of repeating units in the polymer, and ncan vary from 2 to 1000, for example from 2 or 5 to 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700,800 or 900; G₁ and G₂ independently represent a cationic groupcomprising biguanide or guanidine, wherein L₁ and L₂ are directly joinedto a Nitrogen atom of the guanide; L₁ and L₂ are linking groups betweenthe G₁ and G₂ cationic groups in the polymer and independently representan aliphatic group containing C₁-C₁₄₀ carbon atoms, for example an alkylgroup such as methylene, ethylene, propylene, C₄, C₅, C₆, C₇, C₈, C₉ orC₁₀; C₁-C₁₀, -C₂₀, C₃₀, -C₄₀, -C₅₀ -C₆₀, -C₇₀, -C₈₀, -C₉₀, -C₁₀₀, -C₁₁₀,-C₁₂₀, -C₁₃₀ or -C₁₄₀, alkyl; or a C₁-C₁₄₀ (for example C₁, C₂, C₃, C₄,C₅, C₆, C₇, C₈, C₉ or C₁₀, C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀, -C₇₀,-C₈₀, -C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀), cycloaliphatic,heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, or oxyalkyleneradical; or a polyalkylene radical optionally interrupted by one ormore, preferably one, oxygen, nitrogen or sulphur atoms, functionalgroups or saturated or unsaturated cyclic moiety; N and G₃ are optionalend groups; X can be either present or absent; L₃, L₄ and X are linkinggroups between the G₄ and G₅ cationic groups in the polymer andindependently represent an aliphatic group containing C₁-C₁₄₀ carbonatoms, for example an alkyl group such as methylene, ethylene,propylene, C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀-C₆₀, -C₇₀, -C₈₀, -C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀, alkyl; orL₃ and L₄ and X can independently be C₁-C₁₄₀ (for example C₁, C₂, C₃,C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀; C₁-C₁₀, -C₂₀, -C₃₀, -C₄₀, -C₅₀ -C₆₀,-C₇₀, -C₈₀, -C₉₀, -C₁₀₀, -C₁₁₀, -C₁₂₀, -C₁₃₀ or -C₁₄₀), cycloaliphatic,heterocyclic, aromatic, aryl, alkylaryl, arylalkyl, oxyalkyleneradicals; or a polyalkylene radical optionally interrupted by one ormore, preferably one, oxygen, nitrogen or sulphur atoms, functionalgroups as well as saturated or unsaturated cyclic moiety; “G₄” and “G₅”are cationic moieties and can be same or different, and at least one ofthem is a biguanidine moiety or carbamoylguanidine, and the other moietymay be biguanidine or carbamoylguanidine or amine; and cationic moietiesG₄ and G₅ do not contain single guanidine groups; and (ii) an introducedagent as defined in any one of the preceding claims for use in treatinga subject in need of the introduced agent.
 19. (canceled)
 20. A methodof treating a subject in need of an introduced agent comprising the stepof treating the subject with a composition according to claim
 18. 21. Amethod for the detection of an intracellular molecule comprisingdelivering to a cell an entry-promoting agent and an introduced agent asdefined by claim 18, wherein the introduced agent is detectable uponinteraction with the intracellular molecule.
 22. A kit of partscomprising an entry-promoting agent and an introduced agent as definedin claim
 18. 23-26. (canceled)
 27. The method of claim 1 wherein theentry promoting agent and delivered molecule have been mixed orincubated or are provided together in a buffer having a high pH,optionally a pH of 10-13.5.
 28. A method for preparing a complexcomprising an entry promoting agent as defined in any one of thepreceding claims (for example PHMB) and an introduced agent as definedin claim 18, the method comprising mixing or incubating the entrypromoting agent and the introduced agent in a complexation buffer. 29.The kit of claim 22, further comprising a complex wherein the complex isobtainable (or obtained) by a method comprising mixing or incubating theentry promoting agent and the introduced agent in a complexation buffer,for example at a high pH, for example at a pH of 10-13.5.
 30. The methodof claim 28, wherein the method is performed at a high pH, for exampleat a pH of 10-13.5.
 31. The method of claim 21, wherein the introducedagent is a nucleic acid and the method comprises by in situhybridisation, optionally where the nucleic acid is labelled with adetectable molecule.